This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08-DE-104: Genotyping of Existing Cohorts in Craniofacial, Dental and Oral Conditions. Orofacial clefts, specifically cleft lip and cleft palate, are common and costly congenital anomalies whose etiologies remain largely unknown. One of the most promising clues to the causes of orofacial clefts is that women who use vitamins containing folic acid in early pregnancy are at much lower risk for cleft-affected pregnancies. Although the underlying mechanisms by which folic acid contributes to these reduced risks are unknown, evidence suggests that folate intake prevents clefts by compensating for susceptibilities in methyl donor metabolism. However, clear identification of genetic determinants in these pathways through association studies has proven elusive. The approach we propose to further define the molecular genetics mechanisms behind orofacial clefts is based on our observations (Proc Natl Acad Sci, 2008, 105:8055 and an ongoing study) that deep sequencing reveals a substantial amount of low-frequency, nonsynonymous variation in folate pathway genes (frequencies <1%) that has gone unnoticed thus far. Furthermore, a significant fraction of these low-frequency variants affect enzyme function, yet are nutritionally remedial. We hypothesize that genetic susceptibilities in methyl donor metabolism can be etiological for clefts and that these susceptibilities can be conferred by both low-frequency and common variants and by the possible synergy between these. To test this hypothesis, we will sequence the coding regions in 23 methyl donor metabolic genes from a population of 250 cleft-affected infants and 250 controls, for which we also have data on maternal nutritional intake and other epidemiological parameters. We will test all enzyme variants for functional impact and nutritional remediation based on quantitative complementation assays in the yeast S. cerevisiae, and correlate allele distribution and functional studies with clinical phenotype and nutritional data. We hope to better define the causality of orofacial clefts, understand the remedial role of nutritional supplementation, and determine whether additional supplementation may be preventative. This research plan capitalizes on an already existing collaboration that unites a unique combination of expertise for its execution. This proposal should reveal some of the causes of a common form of birth defect known as cleft lip and/or cleft palate. Ultimately, this research may lead to better diagnostic and preventive strategies.